Alzheimer model for drug screening

ABSTRACT

The present invention relates to methods and reagents for drug screening. Screening methods are disclosed employing neuronal cells that have been treated so as to exhibit characteristics associated with a re-entry into the cell cycle. One treatment approach comprises transfection of neuronal cells with an expression vector comprising one or more oncogenes.

FIELD OF THE INVENTION

[0001] The present invention relates to reagents and methods for drugscreening, and more particularly, neuronal cells and tissue forscreening potential Alzheimer therapeutics.

BACKGROUND

[0002] In 1907, Alois Alzheimer described the case of a 51-year-oldwoman with a rapidly degenerating memory who, after a swiftdeterioration, died severely demented four years later. This condition,which now bears Alzheimer's name, describes a fatal degenerativedementing disorder with initial mild memory impairment that progressesunrelentingly to a total debilitating loss of mental and physicalfaculties. Following symptom onset, the course of the disease variesconsiderably from a few years to over 20 years, with a mean survival ofapproximately 8 years. M. A. Smith, “Alzheimer Disease,” Internat. Rev.NeurobioL 42:1 (1998).

[0003] Alzheimer disease affects 10-15% of individuals over 65 years andup to 47% of individuals over the age of 80. In both clinical andautopsy series in the United States and Europe, Alzheimer diseaseaccounts for approximately two-thirds of all dementias affecting elderlyindividuals. D. A. Evans et al., J. Am. Med. Assoc. 262: 2551 (1989).

[0004] The most common and distinctive lesions present with the diseasedbrain are the neuritic senile plaques and neurofibrillary tangles. Themajor protein component of senile plaque cores and vascular amyloid is asmall polypeptide of approximately 4.2 kDa termed amyloid-β. Asignificant fraction of this protein is found to be associated with thecytoskeleton, presumably through its interaction with themicrotubule-associated τ (“tau”) protein. It is believed that theincreased phosphorylated status of tau protein represents one of theearliest neuronal changes prior to the development of neurofibrillarytangles.

[0005] Unfortunately, because of the heterogeneity of the factorsthought to be responsible for Alzheimer disease and the lack of ananimal model displaying the full spectrum of pathological changes,successful pharmacological interventions have not been established. Whatis needed is an easy, reliable method to determine the safety andefficacy of candidate therapeutics for the treatment and/or preventionof Alzheimer disease.

[0006] Definitions

[0007] The term “drug” as used herein, refers to any medicinal substanceused in humans or other animals. Encompassed within this definition arecompound analogs, naturally occurring, synthetic and recombinantpharmaceuticals, hormones, neurotransmitters, etc. The present inventioncontemplates screening test compounds to identify a useful drug for thetreatment of Alzheimers.

[0008] Most current attempts at therapeutics for Alzheimer disease aredirected at neurotransmitter deficiencies. The term “neurotransmitter”includes any compound which functions in the nervous system to result inthe transmission of chemical signals between cells. Examples ofneurotransmitters include, but are not limited to neuropeptides,acetocholine, and amino acids (e.g., GABA). Other compounds are alsocontemplated, including dopamine, norepinephrine, etc.

[0009] The term “GABA” refers to γ-aminobutyric acid, a major inhibitoryneurotransmitter in both vertebrates and invertebrates. Kuffler andEdwards, J. Neurophysiol., 21:589 (1965; Otuska et al., Proc. Natl.Acad. Sci USA 56:1110 (1966); Usherwood and Grundfest, J. Neurophysiol.,28:497 (1965). The term “GABA receptors” thus refers to structuresexpressed by cells and which recognize GABA.

[0010] The present invention contemplates the detection of a variety oftherapeutic compounds, including but not limited to compounds thatinhibit re-entry of neuronal cells into the cell cycle. Such compoundsmay be agonists or antagonists.

[0011] The term “agonist” refers to molecules or compounds which mimicthe action of a “native” or “natural” compound. Agonists may behomologous to these natural compounds in respect to conformation, chargeor other characteristics. Thus, agonists may be recognized by receptorsexpressed on cell surfaces. This recognition may result in physiologicand/or biochemical changes within the cell, such that the cell reacts tothe presence of the agonist in the same manner as if the naturalcompound was present.

[0012] The term “antagonist” refers to molecules or compounds whichinhibit the action of a “native” or “natural” compound. Antagonists mayor may not be homologous to these natural compounds in respect toconformation, charge or other characteristics. Thus, antagonists may berecognized by the same or different receptors that are recognized by anagonist. Antagonists may have allosteric effects which prevent theaction of an agonist. Or, antagonists may prevent the function of theagonist.

[0013] The term “host cell” or “cell” refers to any cell which is usedin any of the screening assays of the present invention. The presentinvention contemplates “host cells” or “cells” in their natural statesas well as genetically altered cells.

[0014] As used in the present invention, the term “transformation”refers to the introduction of foreign genetic material into a cell ororganism. Transformation may be accomplished by any method known whichpermits the successful introduction of nucleic acids into cells andwhich results in the expression of the introduced nucleic acid.“Transformation” includes but is not limited to such methods astransfection, microinjection, electroporation, and lipofection(liposome-mediated gene transfer). Transformation may be accomplishedthrough use of any expression vector.

[0015] The term “gene” refers to a DNA sequence that comprises controland coding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a fill lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity is retained. In the case of the present invention,oncogenes are contemplated and the desired activity is to cause neuronalcells and tissue to exhibit characteristics associated with re-entryinto the cell cycle. Such characteristics may be cellular antigenexpression (e.g. phospho-tau expression). On the other hand, a simplecharacteristic that can be readily measured is the incorporation of anucleic acid precursor (indicating that the cells are traversing theS-phase of the cell cycle). Such precursors include, but are not limitedto, 8-bromodeoxyuridine and tritiated-thymidine.

[0016] The term “wild-type” refers to a gene or gene product which hasthe characteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product. The present inventioncontemplates wild-type oncogenes (e.g. from tumors) as well as oncogenesgenerated by mutation.

[0017] The term “oligonucleotide” as used herein is defined as amolecule comprised of two or more deoxyribonucleotides orribonucleotides, usually more than three (3), and typically more thanten (10) and up to one hundred (100) or more (although preferablybetween twenty and thirty). The exact size will depend on many factors,which in turn depends on the ultimate function or use of theoligonucleotide. The oligonucleotide may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription, ora combination thereof.

[0018] Because mononucleotides are reacted to make oligonucleotides in amanner such that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends.

SUMMARY OF THE INVENTION

[0019] The present invention relates to reagents and methods for drugscreening, and more particularly, neuronal cells and tissue forscreening potential Alzheimer therapeutics. It is not intended that thepresent invention be limited by the nature of the cells employed. In oneembodiment, the cells are primary neurons. It is preferred that the cellemployed is mammalian.

[0020] The present invention contemplates the use of neuronal cellstreated in a manner such that they re-enter the cell cycle. The presentinvention contemplates using these treated cells in compound screeningassays. The neuronal cells may be treated using chemical agents added toculture media. In a preferred embodiment, the neuronal cells are treatedby transfecting them with an expression vector comprising one or moreoncogenes (e.g. a combination of oncogenes).

[0021] In one embodiment, the present invention contemplates a neuronalcell (or cell culture) that has been subjected to treatment so as toexhibit at least one characteristic associated with the re-entry intothe cell cycle. It is not intended that the present invention be limitedby the nature of the treatment. However, in one embodiment, saidtreatment comprises transfection with an expression vector comprisingone or more oncogenes. It is also not intended that the presentinvention be limited by the particular characteristic exhibited.However, in one embodiment, said characteristic comprises increasedphosphorylation of the tau protein.

[0022] In one embodiment, the present invention contemplates a compoundscreening method comprising: a) providing, in any order: i) a reactionvessel; ii) a plurality of neuronal cells treated such that they exhibitat least one characteristic associated with the re-entry into the cellcycle; iii) a test compound b) combining said cells and said compoundwithin said reaction vessel under conditions such that said compound isfree to interact with said cells; and c) detecting said interaction.Again, it is not intended that the present invention be limited by thenature of the treatment. However, in one embodiment, said treatmentcomprises transfection with an expression vector comprising one or moreoncogenes. It is also not intended that the present invention be limitedby the particular characteristic exhibited. However, in one embodiment,said characteristic comprises expression of the tau protein.

[0023] It is not intended that the present invention be limited by thenature of the reaction vessel. Such screening can be done in a microwellof a microtiter plate. Alternatively, newer, high-throughput screeningformats can be used (e.g. silicon-based systems). In one embodiment, thepresent invention also contemplates determining the viability of thecells after contact with the test compound.

DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a schematic illustrating the re-entry into the cellcycle.

[0025]FIG. 2 is a schematic illustrating the control over the cellcycle.

[0026]FIG. 3 is a schematic illustrating interactions between variouscontrolling and inhibiting elements.

[0027]FIG. 4 shows the nucleotide sequences of the coding regions ofcDNAs corresponding to the Ki-ras2 proto-oncogene (cKI-ras2-76) and theKi-ras2 transforming allele present in the human colon carcinoma cellline SW480 (SW-11-1).

[0028]FIG. 5 is a bar graph showing the immunostaining Alzheimer neuronswith antibody directed to SOS-1 and Alz50.

DESCRIPTION OF THE INVENTION

[0029] The present invention relates to reagents and methods for drugscreening, and more particularly, neuronal cells and tissue forscreening potential Alzheimer therapeutics. The present inventioncontemplates the use of neuronal cells treated in a manner such thatthey re-enter the cell cycle. The present invention contemplates usingthese treated cells in compound screening assays. A variety of treatmentmethods can be used, as long as the treatment results in detectablecharacteristics associated with return to the cell cycle. A preferredtreatment method comprises transfecting neuronal cells with anexpression vector comprising the coding sequence of one or moreoncogenes.

[0030] A. Rationale

[0031] Many features of neurons undergoing degenerative changes inAlzheimer disease resemble those of neurons in development that involveentry into the cell cycle. In earlier studies, the present inventorsfound that several markers of the cell cycle, including cyclin dependentkinases and their cognate inhibitors are increased in neurons inAlzheimer disease in comparison to control brain. A. McShea et al.,“Abnormal Expression of the Cell Cycle Regulators P16 and CDK4 inAlheimer's Disease,” Amer. J. Path. 150:1933 (1997). While it may appearparadoxical that cell cycle abnormalities would be important inquiescent neurons, the evidence suggests there is inappropriatestimulation for neurons to re-enter the cell cycle. Indeed, increases inthe levels of cyclin-dependent kinases are the key signal that triggersprogression through the cell cycle. Such cell cycle-related events inpost-mitotic senescent neurons appear to be deleterious and contributeto biochemical abnormalities such as hyper-phosphorylation ofcytoskeletal proteins, apoptotic-like events and oxidative stress, i.e.,the same biochemical abnormalities found in neurons in Alzheimerdisease.

[0032] While the present invention is not limited by any theory ormechanism, as depicted in FIG. 1, it is believed that neuronal cellscharacteristic of Alzheimer disease have initiated by re-entry into thecell cycle. Although abortive exit from the cell division cycle canresult in apoptosis, the continued presence of CDK inhibitors such asp16 and p21 indicate that the cell may not be initiating a mechanismused for programmed cell death but rather entering a dynamic stateassociated with senescence.

[0033] B. Treating Neuronal Cells

[0034] The present invention contemplates the use of neuronal cellstreated in a manner such that they re-enter the cell cycle. The presentinvention contemplates using these treated cells in compound screeningassays. The neuronal cells may be treated using chemical agents added toculture media. The present invention contemplates a method wherebyagents are used to induce nucleotide incorporation in neuronal cells(e.g. primary neurons) with the purpose of producing a population ofneuronal cells that have arrested just prior to mitosis and exhibit aphenotype similar to that observed in Alzheimer's disease

[0035] In a preferred embodiment, the neuronal cells are treated bytransfecting them with constructs comprising one or more oncogenes,thereby driving quiescent neuronal cells out of G0. It is not intendedthat the present invention be limited by the choice of the particularoncogene(s). A variety of oncogenes are contemplated, including but notlimited to naturally occurring oncogenes and oncogenes generated bymutation. Ras Genes. A variety of tumors and tumor cell lines containmutated ras genes, which can be detected by their ability to induce fociof transformed cells upon transfection of monolayer cultures of NIH 3T3mouse fibroblasts. Several of these activated ras genes have been clonedand found to arise by mutation of normal cellular proto-oncogenes. Insome cases, the alterations responsible for the activation of theseoncogenes are point mutations that affect the protein-encoding portionsof these genes and change amino acid 12 or 61 of the 21-kilodalton rasprotein (“p21”). The normal cellular Harvey sarcoma virus (Ha), Kirstensarcoma virus (Ki), and N-ras proteins carry glycine as residue 12.Spontaneous mutations causing replacement of Gly 12 by a variety ofresidues, including valine, aspartic acid, or cysteine, have been shownto convert the human Harvey and Kirsten virus genes into activeoncogenes. See e.g. M. McCoy et. al., “Human Colon Carcinoma Ki-ras2Oncogene and Its Corresponding Proto-Oncogene,” Mol. Cell. Biol. 4:1577(1984) (FIG. 4 of the present application was obtained from thisreference). Myc Genes. Myc is a nuclear proto-oncogene, and is thecellular counterpart to the oncogene v-myc carried by anacutely-transforming strain of the retrovirus avian leukosis virus. Mycis involved in the chromosome translocation t(8:13)(q24:q32) found inBurkitt's lymphoma where it is translocated into the immunoglobulinheavy chain gene. It encodes a transcription factor, forming aDNA-binding hetero-oligomer with the transcription factor Max. Severalmyc-related genes include c-myc, L-myc, and N-myc (Winters et al.,Cancer, Principles & Practice of Oncology 3rd edition, pp. 59-60, J. B.Lippincott Company [1989]).

[0036] The c-myc gene product is a nuclear protein expressed in a widevariety of cell types, and has been implicated in the control of normalcell growth as well as transformation, but its exact function in unknown(Bentley and Groudine, Nature 321: 702-706 [1986]). The c-myc regionswere initially defined by their homology with thetransformation-specific segment (v-myc) of the avian defective acuteleukemia virus MC29. In this virus, myc sequences interrupt and displaceretroviral structural gene regions. This results in novel viral RNA andprotein products composed of viral structural sequences covalentlylinked to v-myc (Hann et al., Cell 34: 789-798 [1983]). Examples ofhuman myc oncogene GenBank accession numbers are M12026, M12027 andM16261.

[0037] Combinations Of Oncogenes. Data from the group of Nevins hasdemonstrated that certain combinations of oncogenes (Myc and Ras) can beused to drive quiescent fibroblasts out of G0, through G1, and intoS-phase. Leone et al., “Myc and Ras collaborate in inducing accumulationof active cyclin E/Cdk2 and E2F” Nature 387:422 (1997). The presentinvention contemplates using such combinations of oncogenes to inducecell-cycle changes in neuronal cells and tissue. In one embodiment, thepresent inventors have adapted the adenovirus vector delivery system toinduce high level expression of a constitutively active form of ras andwild-type c-myc into neuronal cells, such as primary neuronal cultures(described in the experimental section below). This transfection isadequate to induce alterations in cytoskeletal phosphorylation in adultneurons akin to that that occurs in Alzheimer disease. Thus, the presentinvention provides an in vitro model of the diseased brain which readilypermits drug screening.

[0038] Other approaches. The present invention also contemplates otherapproaches to achieve the above-described effect. Other approachessimilar to viral ras stimulation include constitutively active forms of:MAP kinase. Son-of-sevenless 1, GRB2, SHC, growth factor receptorsincluding the TRK and NGF families. Alteratives to myc that arecontemplated include mutants of mad and max.

[0039] The present invention also contemplates approaches involvingtranscription factor overexpression, e.g. E2F family memberoverexpression, modulation of retinoblastoma gene expression (e.g. byviral gene products), overexpression of cyclin dependent kinase familymembers or expression of cyclin dependent kinase inhibitor motifs topromote re-entry into the cell cycle by permitting reactivation ofkinase activity pertaining to cell cycle control.

[0040] C. Detecting Re-Entry into the Cell Cycle

[0041] Events central to growth and development such as cell division,differentiation and quiescence are under tight regulation (FIG. 2).Cyclins and cyclin-dependent kinases form complexes that are able tophospho-regulate a wide variety of substrates involved in the orderlyprogression through the mitotic cycle (FIG. 3). Cells receiving externalgrowth stimulus upregulate cyclin-dependent kinases and their cognateactivating cyclins to orchestrate DNA replication, cytoskeletalre-organization and cellular metabolism required for proliferation.Whereas progression through the cell division cycle is controlled inpart by the sequential synthesis and degradation of cyclins, growtharrest or terminal differentiation are controlled initially bycyclin-dependent kinase inhibitors, including p16, p21, p27 and p57which, among other functions, directly inhibit the cyclin-CDK complex(FIG. 3). As depicted in FIG. 2, transition from the G1 phase of thecell cycle and commitment to S phase/DNA replication is thought to bemediated by the association of G1 cyclin/CDK complexes such asCDK2/cyclin E and CDK4/D-type cyclins. Phosphorylation of theretinoblastoma protein by CDK4/cyclinD, for example, enables release ofE2F transcription factor and expression of genes required for DNAreplication and proliferation. Active CDK complexes are negativelyregulated by the competitive binding of tumor suppressor gene productssuch as p16, which disrupt the complex and indirectly inhibit E2Fmediated gene expression.

[0042] The present invention contemplates detecting cell cycle-relatedproteins that are upregulated (as a result of re-entry into the cellcycle) as well as their relationship, both temporal and spatial, to moreestablished markers of the disease. The present invention provides ameans for detecting cell cycle abnormalities in the neuronal death thatare characteristic of Alzheimer disease.

[0043] Neurons normally develop to exit from the mitotic cell cycle andenter into a quiescent state equivalent to G0. In Alzheimer disease,however, neurons express markers common to cells in a variety of stagesof the cell cycle.

[0044] Using the transfected primary neurons discussed above,alterations in cytoskeletal phosphorylation has been detected that isakin to that found in neurons in Alzheimer disease. Detection is readilyperformed using immunocytochemical and immunoblotting analysis of cellcycle markers. In this regard, it is helpful to group markers groupedaccording to cell cycle position. For example, progression from G0(Cyclin D, Cyclin E, Cyclin A, PCNA, Rb phosphorylation, E2f);progression into G2 (Cyclin B); exit from mitosis (p16, p14-arf, p19,p21, p27, p57). The spatio-temporal distribution and expression of thesecell cycle antigens can also be correlated with that of other knownmarkers of disease such as oxidative, mitochondrial, apoptotic, andcytoskeletal abnormalities.

[0045] D. Drug Screening Assay Systems

[0046] The screening assays are contemplated as utilizing variousneuronal cells. In one embodiment, the present invention contemplatesprimary neurons that have been transfected (transiently or stably) withan expression vector comprising a combination of oncogenes. Importantly,the use of potential agonists or antagonists in these assay systemsshould allow for the detection of new compounds (e.g., drugs) whichovercome or inhibit deleterious cell-cycle-related events.

[0047] The in vitro system permits the screening of many compounds.Importantly, the method is amenable to automation, providing thebenefits of high throughput and reducing the amount of personnel timenecessary to perform the assay.

EXPERIMENTAL

[0048] The following examples are provided in order to demonstrate andfurther illustrate certain aspects of the present invention. Theexamples are not to be construed as limiting the invention.

[0049] In some of the experiments described below, immunocytochemistryis performed. In some cases comparisons are made with brain tissue fromdiseased individuals. The methods involve obtaining tissue, fixation andstaining.

[0050] Tissue: Brain tissue is obtained at autopsy from clinically andpathologically confirmed cases of AD by using NIA and CERAD criteria)and compared with tissue from non-demented control cases with similarpostmortem intervals prior to fixation.

[0051] Fixation: Tissues are fixed in methacarn (methanol: chloroform:acetic acid, 6:3:1) or formalin for 16 hr at 4° C., dehydrated throughgraded ethanol followed by xylene, and embedded in paraffin. Six-micronthick sections are cut and mounted on Silane (Sigma, St. Louis)-coatedstandard glass microscope slides for in situ hybridization andimmunocytochemical use.

[0052] Immunocytochemistry: Immunostaining is performed using theperoxidase-antiperoxidase procedure. After deparaffinization in xyleneand rehydration through graded ethanol, endogeneous peroxidase activityis inhibited by 20 min incubation in 3% H₂O₂ and nonspecific proteinbinding sites are blocked with 10% normal goat serum in Tris-bufferedsaline (150 mM Tris-HCl, 10 mM NaCl, pH 7.6).

[0053] Immunostaining is readily achieved by theperoxidase-antiperoxidase technique using 3,3′-diaminobenzidine aschromogen. Adjacent sections are immunostained with a variety ofantibodies (e.g. available from commercial sources such as UBI, OncogeneScience, Santa Cruz, Calif.) to confirm either the identity and locationof pathological structures or the presence of various diseaseparameters. In certain cases, double stain experiments are used wherethe second antigen is localized with the alkaline phosphatase techniquewith fast blue as substrate. Alternatively, sequential stains are usedwhere a soluble chromagen is employed.

[0054] Adsorption experiments can be performed to confirm thespecificity of antibody binding. The immunostaining protocol isrepeated, except here using adsorbed antiserum in parallel. Adsorbedantisera can be generated by incubation of primary antisera withpurified protein diluted to a final concentration of 1-100 (g/ml for 3hr at 37° C. Adsorption of anti-tau antibody can be performed as acontrol against artifactual absorption.

Example 1

[0055] In this example, components known to trigger cellularproliferation and differentiation, upstream of the ras/mitogen-activatedkinase pathway, were investigated. FIG. 5 shows the quantitative doubleimmunolabeling results with both SOS-1 and Alz50 in Alzheimer neurons.Three cases showed that the vast majority of neurons labeled with bothantisera or with SOS-1 exclusively, indicating that SOS-1 isfunctionally significant in the disease process.

Example 2

[0056] The present invention contemplates the transfection of neuronalcells, thereby treating the cells so that they exhibit characteristicsassociated with the re-entry into the cell cycle. The present exampledescribes one embodiment of a construct comprising oncogenes andsubsequent transfection of primary neurons.

[0057] Design Of The Construct. C. Farnsworth and L. Reig describe thecreation of constructs comprising a coding sequence of a ras oncogene.See C. Farnsworth and L. Reig, Mol. Cell. Biol. 11: 4822 (1991).Briefly, the gene described by those authors comprised the 5′ end ofviral ras^(H) which was converted to the coding sequence of cellularras^(H) by site-directed mutagenesis, and the 3′ end of a cellular rascDNA clone. An approximately 900 bp HindIII-BamHI fragment of the rasgene was excised from M13 double-stranded DNA and inserted in place ofthe comparable fragment removed from the bacterial expression vectorpXCR The 0.7-kb Bg/II-BamHI fragment of pXCR was subcloned into theBamHI site of the mammalian expression vector pZIPneoSV(X).

[0058] For the present experiment, Ad-Ras^(N17) was constructed byligation of the HindIII/XbaI fragment from pRsαRas^(N17) of Fransworthand Reig and the BamHI fragment from pZIP-NeoSV(x) into the similarlydigested pGEM-CMV vector. Ad-Myc was constructed by ligation of theHingIII/XbaI fragment from CMV-Myc containing the mouse c-myc cDNA intosimilarly digested pGEM-CMV vector. Viral stocks were created and viruswas purified. Viral titres are determined by an indirectimmunofluorescent assay specific for the viral 72K E2 gene product anddefined as focus forming units (FFU) per ml.

[0059] Transfection of Primary Neurons. Primary neurons are preparedaccording to standard laboratory protocol. Briefly, newborn mice aredecapitated and the brain removed. The meninges are removed and thetissue minced followed by dissociation of the cells in 0.05% trypsin(Type III, Sigma Chemical, St. Louis) at 37° C. for 20 minutes. The cellsuspension is triturated in 4 Kunitz unit/ml DNase I and then plated onpoly-L-lysine coated Falcon flasks. Shaking the flask 4-6 hours afterplating at which time only the astrocytes are strongly adherent canisolate astrocytes. Neurons can be isolated by gently shaking 4-6 hoursafter plating the cortical cultures which detaches the neurons from theflask. The neurons are recovered in the media and replated onpoly-L-lysine coated plates. Finally, microglial cells can be isolatedby shaking cortical cultures on an orbital shaker 10 days after platingat 180 rpm for 1 hour after which they are plated on poly-L-lysinecoated plates for one hour and the media containing other cell types isremoved. The cell cultures can be assessed for purity using standardimmunological techniques. The next day, the cells are washed once withD-MEM and the culture medium replaced with D-MEM containing 0.25% serumand cells further incubated for 48 h before virus infection. Cells onplates are infected in D-MEM with 20 mM HEPES, pH 7.2, for 75 min at 37°C. at a cell-to-volume ratio of 0.5×10⁶ ml⁻¹. After infection, fourvolumes of 0.25% serum/D-MEM are added to each plate and the cellsincubated at 37° C.

Example 2

[0060] This example describes the changes detectable after thetransfection of cells according to the procedures describe above inExample 1. Having adapted the adenovirus vector delivery system toinduce high level expression of a constitutively active form of ras andwild-type c-myc into primary hippocampal neurons, the neurons werecultured and stained. The results showed that the neurons persistin-culture with an enlarged cell body and are immunopositive forphospho-tau (immunolabeled with AT8). The results indicate thatphosphorylated tau epitopes are increased in primary neuronal culturestransfected with Ras/Myc in comparison to those transfected with ras orMyc alone or non-transfected/mock transfected controls (cells were fixed24 hours post-transfection).

[0061] From the above descriptions and examples, it should be clear thatthe methods and reagents of the present invention represent an easy,reliable method to determine the safety and efficacy of new compoundsfor the treatment of Alzheimer disease.

What is claimed is:
 1. A neuronal cell that has been subjected totreatment so as to exhibit at least one characteristic associated withthe re-entry into the cell cycle.
 2. The neuronal cell of claim 1,wherein said treatment comprises transfection with an expression vectorcomprising one or more oncogenes.
 3. The neuronal cell of claim 1,wherein said characteristic comprises increased phosphorylation of thetau protein.
 4. The neuronal cell of claim 1, wherein said cell ismammalian.
 5. A compound screening method comprising: a) providing, inany order: i) a reaction vessel; ii) a plurality of neuronal cellstreated such that they exhibit at least one characteristic associatedwith the re-entry into the cell cycle; and iii) a test compound; b)combining said cells and said test compound within said reaction vesselunder conditions such that said compound is free to interact with saidcells; and c) detecting said interaction.
 6. The screening method ofclaim 5, wherein said treatment comprises transfection with anexpression vector comprising one or more oncogenes.
 7. The screeningmethod of claim 5, wherein said characteristic comprises expression ofthe tau protein.
 8. The screening method of claim 5, wherein said cellsare mammalian cells.
 9. The screening method of claim 5, furthercomprising the step of determining the viability of said cells aftercontact with said test compound.